Systems and methods of all-optical fourier phase contrast imaging using dye doped liquid crystals

ABSTRACT

An assembly for converting a microscope into a phase contrast microscope includes a first optical Fourier element that Fourier transforms light from a coherent light source, a cell in the Fourier plane arranged to receive light from the first optical Fourier element, a second optical Fourier element arranged to receive light from the cell and inversely Fourier transform the received light to provide an image, an image sensor that detects the image and generates an electronic representation of the image, and an adaptor capable of coupling the first and second Fourier elements, the cell, and the image sensor to the microscope such that the first Fourier element Fourier transforms light collected by the microscope objective. The cell includes liquid crystal molecules having a phase transition temperature, wherein at temperatures exceeding the phase transition temperature, light transmitting through the liquid crystal molecules obtains a different phase than light transmitting through the liquid crystal molecules at temperatures below the phase transition temperature.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of and claims the benefit under 35U.S.C. §121 of U.S. patent application Ser. No. 11/935,910, filed Nov.6, 2007 and entitled “Systems and Methods of All-Optical Fourier PhaseContrast Imaging Using Dye Doped Liquid Crystals,” which claims thebenefit under 35 U.S.C. §119(e) of U.S. Provisional Application No.60/856,972, filed Nov. 6, 2006 and entitled “Phase Contrast ImagingUsing Dye Doped Liquid Crystals,” the entire contents of bothapplications are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This research is supported in part by a BAA contract W911QY-04-C-0063from U.S. Army Natick Soldier Center.

BACKGROUND

1. Field

The disclosed subject matter generally relates to phase contrastimaging, e.g., phase contrast microscopy.

2. Discussion of Related Art

Translucent objects or phase objects can alter only the phase of theoptical wave, not its amplitude. Hence, these objects are very difficultto see with the naked eye and cannot be captured by an ordinary camera.A phase contrast microscope can be used to obtain high-contrast imagesof transparent specimens, such as living cells (usually in culture),micro-organisms, thin tissue slices, lithographic patterns, fibers,latex dispersions, glass fragments, and subcellular particles (includingnuclei and other organelles). One useful feature of a phase contrastmicroscope is that living cells can be examined in their natural statewithout being fixed, and/or stained. As a result, the dynamics ofongoing biological processes can be observed and recorded in highcontrast with sharp clarity of minute specimen details.

In 1933, Zernike developed a non-destructive mechanism based on theprinciple of phase contrast to observe translucent microscopic objects.It is a two step process: (1) separation of deviated and undeviatedcomponents in the light transmitted through the specimen with a π/2phase difference between them and (2) obtaining an additional π/2 phaseseparation thereby converting phase information into amplitude(intensity) contrast for display. If the undeviated light is phaseshifted by π/2, then the undeviated and diffracted light arriving at theeyepiece would produce destructive interference and the object detailsappear dark in lighter background. This is known as dark or positivephase contrast. If, however, the undeviated light is phase shifted by−π/2 then the diffracted and undeviated light beams interfereconstructively. This produces a bright image of the details of thespecimen in dark background and is known as negative or bright contrast.This principle is exploited for the phase contrast microscope.

Existing phase contrast microscopes employ a tungsten-halogen lamp as alight source and a condenser annulus for separation of the deviated andundeviated light. They also use phase plates for generating theadditional phase retardation between undeviated light and lightdiffracted by the object, thereby transforming minute variations inphase of the object into corresponding changes in image contrast. Thecollimated light passes through the condenser plate which typicallycontains several transparent annular rings (carefully positioned anddesigned to be an optical conjugate to a phase plate residing in theimage plane) and is focused onto the specimen. The light transmitted bythe specimen consists of undeviated light and diffracted light. Theundeviated and diffracted light differs in phase by π/2 due to theinherent phase variations in the specimen. The light is then collectedby the objective and is spatially separated at its back focal plane. Aphase plate selectively placed at this back focal plane introduces anadditional π/2 relative phase difference. Thus the undeviated anddiffracted light interferes destructively so that the phase variationsin the specimen appear bright against a dark background. Two types ofphase plates, positive and negative, are available to produce a brightimage in dark background or vice versa.

However, there are some unavoidable disadvantages associated with theuse of these plates:

-   -   1. Halo and shade-off contrast patterns are frequently observed        in phase contrast images. These observed intensity patterns do        not directly correspond to the optical path difference between        the specimen and the surrounding medium. The artifacts depend on        both the geometrical and optical properties of the phase plate        and the specimen being examined. In particular, the width and        transmittance of the phase plate material play a critical role        in controlling these effects. In addition, these effects are        heavily influenced by the objective magnification. Apodized        phase plates are used for reducing the severity of halos.    -   2. In order to resolve minute details and edges in the specimen,        a large angle of diffracted light must be captured by the        microscope objective and must be brought into a sharp focus at        the image plane. The condenser aperture diaphragm opening size        partially controls the coherence of the light incident on the        specimen. Decreasing the opening size of the diaphragm yields        greater spatial coherence but it introduces diffraction related        artifacts. Thus the system is limited by the working numerical        aperture of the objective thereby reducing the resolution of the        instrument.    -   3. When the object is changed or a different magnification is        desired, the bright-field image has to be obtained first and        then the condenser plate has to be rotated to position the        annular ring to match the new phase plate. Thus as a result of        frequent rotations of the condenser plate, the annular ring        tends to be out of alignment with the phase plate requiring        regular maintenance of the system. Special tools are provided        for adjusting the condenser plate, which require skill and        experience on the part of the operator. Furthermore, rotation of        the condenser plate can sometimes cause the specimen to move as        it is positioned just before the condenser.

Existing phase contrast microscopes have been modified since theirinvention, in terms of phase plate design and detection schemes.However, conventional phase contrast microscopes do not exploitadvantages that come with a coherent source. For example, the whitelight sources of conventional phase contrast microscopes cannot provideFourier transformation, as a result which the object information cannotbe well separated at the Fourier plane.

With the growing demand for a variety of imaging modalities that offerdifferent distinct advantages, improved methods for imaging phaseobjects in transparent media and imaging phase objects in tissue-likescattering media are needed.

SUMMARY

Embodiments of the invention provide systems and methods of all-opticalFourier phase contrast imaging using dye doped liquid crystals.

Under one aspect, a phase contrast imaging system includes a coherentlight source emitting a coherent beam, the beam being directed toward asample area; a lens arranged to collect at least part of the beam fromthe sample area; a first optical Fourier element that Fourier transformsthe collected beam, wherein the Fourier transform occurs in a Fourierplane; a liquid crystal cell in the Fourier plane that transmits atleast part of the transformed beam, wherein the cell includes liquidcrystal molecules having a phase transition temperature, and wherein attemperatures exceeding the phase transition temperature, lighttransmitted through the liquid crystal molecules obtains a differentphase than light transmitted through the liquid crystal moleculesobtains at temperatures below the phase transition temperature; a secondoptical Fourier element that receives the transmitted beam and inverselyFourier transforms the transmitted beam to provide an image; an imagesensor that detects the image and generates an electronic representationof the image; and an optical element configured and arranged to adjustthe beam intensity to a level at which part of the transformed beam hasan intensity sufficient to heat a portion of the liquid crystalmolecules above the phase transition temperature.

Some embodiments include one or more of the following features. The lensincludes a microscope objective. The optical element selected to adjustthe beam intensity includes a neutral density filter. The first andsecond optical Fourier elements include lenses. The image sensorincludes a CCD. A polarizer positioned between the second opticalFourier element and the image sensor, the polarizer being rotatable to aposition selected to eliminate at least a part of the information aboutthe sample area from the image. An optical element to direct at leastpart of the beam toward a fluorescence imaging system. At temperaturesbelow the phase transition temperature, the liquid crystal molecules arebirefringent, and wherein at temperatures above the phase transitiontemperature, the liquid crystal molecules are isotropic. The beam has apredominant polarization, and wherein the liquid crystal cell isoriented at about 45° to the predominant polarization. The liquidcrystal cell further includes dye molecules selected to at leastpartially absorb the beam, and wherein at least partial absorption ofthe beam by a portion of the dye molecules heats the part of the liquidcrystal molecules above the phase transition temperature. The phasetransition temperature, the liquid crystal molecules are in an alignednematic phase. The optical element is configured and arranged to adjustthe beam intensity to a level at which a portion of the transformed beamtransmits through the cell with a phase that is delayed relative to another portion of the transformed beam by one of about π/2 and about−π/2. The portion of the transformed beam that is phase delayedcorresponds to low spatial frequencies, and wherein the other portion ofthe transformed beam corresponds to spatial frequencies that are higherthan the portion that is phase delayed and also has a lower intensitythan does the portion that is phase delayed. The coherent light sourceincludes a continuous-wave laser. The coherent light source includes adiode. The liquid crystal cell is passive, in that no voltage is appliedto the cell. The optical element is further configured and arranged toadjust the beam to an intensity such that it does not damage a livingorganism placed in the sample area.

Under another aspect, a method of imaging an object includes generatinga coherent beam; irradiating an object with the coherent beam;collecting at least a part of the beam that irradiated the object;Fourier transforming the collected beam; phase delaying a portion of thetransformed beam relative to another portion of the transformed beam;inversely Fourier transforming the partially phased-delayed beam; anddetecting the inversely Fourier transformed beam.

Some embodiments include one or more of the following features. Fouriertransforming the collected beam includes transmitting the collected beamthrough a lens. Inversely Fourier transforming the partiallyphase-delayed beam includes transmitting the partially phase-delayedbeam through a lens. Phase delaying the portion of the transformed beamrelative to another portion of the transformed beam includestransmitting the transformed beam through a cell including liquidcrystal molecules. The liquid crystals have a phase transitiontemperature, and wherein at temperatures exceeding the phase transitiontemperature, light transmitting through the liquid crystal moleculesobtains a different phase than light transmitting through the liquidcrystal molecules obtains at temperatures below the phase transitiontemperature. Selecting an intensity of the beam such that a portion ofthe transformed beam heats the liquid crystal molecules to a temperatureexceeding the phase transition temperature, and another portion of thetransformed beam does not heat the liquid crystal molecules to atemperature exceeding the phase transition temperature. Selecting theintensity of the beam such that the portion of the transformed beam thatheats the liquid crystal molecules to a temperature exceeding the phasetransition temperature accrues a phase delay of one of about π/2 and−π/2 relative to the portion of the transformed beam that does not heatthe liquid crystal molecules to a temperature exceeding the phasetransition temperature. Below the phase transition temperature, theliquid crystal molecules are in an aligned nematic phase. The cellfurther includes a dye selected to absorb a wavelength of the beam. Thecell is passive in that no voltage is applied to the cell. The objectincludes a living organism that is not damaged by the coherent beam.Detecting the inversely Fourier transformed beam includes irradiating animaging device with the inversely Fourier transformed beam. Displayingoutput of the imaging device on a display device.

Under another aspect, an assembly for converting a conventionalmicroscope into a phase contrast microscope, wherein the conventionalmicroscope includes a microscope objective and a specimen stage, and themicroscope objective is constructed and arranged to collect lightdirected toward the specimen stage, includes a first optical Fourierelement that Fourier transforms light, wherein the Fourier transformoccurs in a Fourier plane; a cell in the Fourier plane, wherein the cellincludes liquid crystal molecules having a phase transition temperature,and wherein at temperatures exceeding the phase transition temperature,light transmitting through the liquid crystal molecules obtains adifferent phase than light transmitting through the liquid crystalmolecules obtains at temperatures below the phase transitiontemperature; a second optical Fourier element that receives thetransmitted beam and inversely Fourier transforms the transmitted beamto provide an image; an image sensor that detects the image andgenerates an electronic representation of the image; and an adaptorcapable of coupling the first and second Fourier elements, the cell, andthe image sensor to the conventional microscope such that the firstFourier element Fourier transforms light collected by the microscopeobjective.

In some embodiments, the microscope includes a brightfield or ordinarymicroscope.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A is a schematic illustration of a Fourier phase contrast imagingsystem, according to some embodiments of the invention.

FIG. 1B schematically illustrates the mechanism of the phase delaysaccumulated by light transmitting through different parts of the Fourierphase contrast imaging system of FIG. 1A, according to some embodimentsof the invention.

FIG. 2A is an image of an amoeba obtained using the system of FIG. 1A inbright-field microscopy mode (with low laser power), according to someembodiments of the invention.

FIG. 2B is a phase contrast image of an amoeba obtained using aconventional phase contrast microscope.

FIG. 2C is a phase contrast image of the amoeba of FIG. 2A obtainedusing the system of FIG. 1A in Fourier phase contrast imaging mode (withincreased laser power), according to some embodiments of the invention.

FIG. 3A is an image of a live paramecium obtained using the system ofFIG. 1A in bright-field microscopy mode, according to some embodimentsof the invention.

FIG. 3B is a phase contrast image of a live paramecium obtained using aconventional phase contrast microscope.

FIG. 3C is a phase contrast image of the live paramecium of FIG. 3Aobtained using the system of FIG. 1A in Fourier phase contrast imagingmode, according to some embodiments of the invention.

FIG. 4A is an image of onion cells in a scattering medium obtained usingthe system of FIG. 1 in bright-field microscopy mode, according to someembodiments of the invention.

FIG. 4B is an image of the onion cells of FIG. 4A obtained using thesystem of FIG. 1A in Fourier phase contrast imaging mode, according tosome embodiments of the invention.

FIG. 5A is an image of a glass speck placed on a glass micro slideobtained using the system of FIG. 1A in bright-field microscopy mode,according to some embodiments of the invention.

FIG. 5B is an image of the glass speck of FIG. 5A obtained using thesystem of FIG. 1A in Fourier phase contrast imaging mode, according tosome embodiments of the invention.

FIG. 5C is a negative phase-contrast image of the glass speck of FIG. 5Aobtained using the system of FIG. 1A in Fourier phase contrast imagingmode, according to some embodiments of the invention.

FIG. 6 is a schematic illustration of a conventional ordinarymicroscope.

FIG. 7 is a schematic illustration of a microscope that is modified toperform phase contrast microscopy, according to some embodiments of theinvention.

FIG. 8 is a schematic illustration of the system of FIG. 1A that hasbeen modified to further provide perform fluorescence imaging of asample, according to some embodiments.

DETAILED DESCRIPTION

Embodiments of the invention are directed to systems and methods ofall-optical Fourier phase contrast imaging using a low power coherentsource (laser) and dye-doped liquid crystals. In general, the Fourierspectrum of an object contains low spatial frequencies at the center ofthe spectrum, with high intensities, while high spatial frequencies areon the edges, with lower intensities. The laser source provides preciseseparation of these frequency regimes through an all-optical Fouriertransform. In some embodiments, high monochromaticity of the coherentsource facilitates a well defined Fourier plane in which differentspatial frequency bands are clearly resolved. In addition the intensityof the laser source makes object features bright and clearly visible.Some embodiments provide bright-field, positive phase contrast andnegative phase contrast images of “phase objects,” i.e., objects thatare at least partially optically transparent and thus are difficult toimage using conventional amplitude-based imaging such as, for example,ordinary bright-field microscopes and photographs. Specifically,different regions of a phase object have different opticalcharacteristics, for example, have different indices of refractionand/or different thicknesses, which diffract, refract, and/or impartphase changes onto coherent light passing through the object relative tolight that does not pass through the object. The difference in phasebetween light that passes through the object, and that does not passthrough the object, is manipulated using a dye-doped liquid crystal cellin the Fourier plane of the object, as described in greater detailbelow. The resulting phase difference is used to generate a phasecontrast image of the object. While conventional phase contrastmicroscopy uses a white-light source and a phase plate (fixed in theamount of phase retardation it can induce and diameter), the systems andmethods described herein are robust and “self-adaptive,” that is,readily provide images regardless of changes in the shape, size andmagnitude of phase variations of phase objects. The systems and methodsare also relatively user-friendly, allowing the contrast of images of aphase object to be modified by simply changing the intensity of thelight that impinges the sample. As illustrated below, the systems andmethods can be used to produce high-quality phase contrast images of aphase object (even in a scattering medium). For example, the shape ofmicro organisms can be clearly displayed and quantitative informationsuch as the dimensions of the objects can be obtained.

In many embodiments, coherent light waves that are in phase with oneanother are directed in phase onto an at least partially transparentobject. Some of the light waves accumulate a phase shift as they passthrough the object, while light waves that do not pass through theobject do not accumulate a phase shift. The light is then Fouriertransformed using a lens or microscope objective, and a cell containingdye-doped liquid crystals is placed at the resulting Fourier plane. Thedye in the cell at least partially absorbs the light, and the resultingtemperature increase causes an intensity-dependent, liquid-crystal phasetransition within the cell. The spatial profile of the temperatureincrease corresponds to the spatially-varying intensity of the Fouriertransform of the object. In some regions of the liquid crystal, thelight intensity (and concomitant temperature increase) is sufficientlyhigh to cause the liquid crystal molecules in those regions to changephase, for example, to an isotropic phase. In other regions of theliquid crystal, the light intensity is insufficiently high to cause theliquid crystal molecules in those regions to change phase. Theparticular phase of the liquid crystal modifies the phase of the lightpassing through the cell. The phase of the liquid crystal (andconcomitant relative phase shift of different regions of the Fouriertransform of the light) can be modified by adjusting the amplitude ofthe light with which the sample is irradiated.

A phase-contrast image of the object is then obtained by detecting phasedifferences between the high and low spatial frequencies, e.g., byinterfering the high and low spatial frequencies with each other. Insome embodiments, this is done by inversely Fourier-transforming thelight transmitted by the cell, and then imaging the light onto a CCDarray. At the CCD (i.e., in the image plane of the light), the differentspatial frequencies of the light interfere with one another, generatingan amplitude image of the object that is based, in part, on the relativephases that the object imparts on the light, as well as on the relativephases that the liquid crystal cell imparts on the light. The contrastof the image can be modified by adjusting the amplitude of the light. Inone example, the amplitude of the light is selected to generate anapproximately π/2 or −π/2 phase difference between the high and lowspatial frequencies.

The CCD array can be, for example, a two-dimensional array of detectorsintegrated into single, compact electronic chip. The CCD array convertsphotons to electrons using closely spaced metal-oxide-semiconductor(MOS) diodes and thereby generates a discrete electronic representationof a received optical image. A controller/processor reads the imagerepresentation from the CCD sensor pixel-by-pixel and organizes it intoa digital array. The digital array can then be output to a memory orimage store. The images can be displayed on an image display, such as acathode ray tube or another type of electronic image display.

Some embodiments include a nematic liquid crystal cell in the plane ofthe Fourier transform of the light, e.g., a cell containing twistednematic liquid crystals, and an absorber or dye that is selected to atleast partially absorb the wavelength of interest, and to cause asufficient temperature increase in the liquid crystal upon irradiationto induce a phase change in the liquid crystal. Nematic liquid crystals(LC) include rod-like molecules which line up parallel to a preferreddirection and hence are anisotropic. When a linearly polarizedmonochromatic light wave propagates through a homogeneously aligned LCcell with its polarization axis at 45° to the axis of orientation, theanisotropy property of the liquid crystal adds a certain amount of phaseto the transmitting beam. This phase is attributed to the refractiveindex differences of the ordinary and extraordinary rays.

Relatively high intensity regions of the Fourier transform of the light,e.g., low spatial frequencies at the center of the Fourier spectrum, areintense enough to cause molecules in those regions to undergo atransition from nematic or anisotropic phase to isotropic phase. Lowerintensity regions of the Fourier transform of the light, e.g., highspatial frequencies near the edges of the Fourier spectrum, are notsufficiently intense to induce a phase transition, and molecules inthese regions remain in an anisotropic phase. Aligned liquid crystalmolecules (molecules that are in anisotropic or nematic phase) add acertain amount of phase to the incident light wave as it passes through,whereas isotropic liquid crystals substantially do not add additionalphase to the transmitted beam. Thus the high intensity, low spatialfrequency light will transmit through the self-induced isotropic phaseof liquid crystal cell without accumulating phase change, while the lowintensity, high spatial frequency light will acquire a phase changerelative to the high intensity light as it transmits through the liquidcrystal phase (anisotropic phase) of the liquid crystal cell. This leadsto a relative phase difference between these two spatial frequencyregions, which is then used to generate a phase contrast image.Usefully, the phase difference is on the order of about π/2 or −π/2,which generates images of high contrast. However, other phasedifferences also produce useful images. In many embodiments, the liquidcrystal cell is passive, that is, it needs no applied voltage in orderto perform its function.

The relative phase retardation experience by light transmitting throughthe cell is expressed by Γ=πΔnd/λ, where d is the cell thickness, λ isthe wavelength, and Δn=(n_(e)−n₀) is the induced birefringence. As thetemperature of the liquid crystal increases, the ordinary refractiveindex (n₀) increases while the extraordinary refractive index (n_(e))decreases. Thus, the birefringence decreases with increasing temperatureand vanishes when the liquid crystal molecules undergo phase transition,liquid crystal phase to isotropic phase. At low light input intensities,the temperature of the liquid crystal is well below its phase transitiontemperature T_(c). Thus, a phase, e.g., of 90°, is added to thetransmitted beam because of the large birefringence Δn in the liquidcrystal phase. When the incident light intensity increases, thetemperature of the liquid crystal increases owing to the absorption bydye molecules. At temperatures exceeding the phase transitiontemperature of the liquid crystals (T≧T_(c)) there is no birefringenceand hence light transmitting through those crystals experiences no phaseretardation. This results in the increase of ordinary refractive index(n₀) and decrease in the extraordinary refractive index (n_(e)). ForT≧T_(c),n₀=n_(e) and the induced birefringence Δn vanishes. Hence noadditional phase is added to the transmitted beam. Therefore, if twolight beams of different intensity are incident simultaneously atdifferent spatial locations on the liquid crystal, the local liquidcrystal molecules undergo respective intensity-dependent, liquid-crystalphase transitions. This leads to a relative phase difference, e.g., ofπ/2, −π/2, or some other value, between these two light beams at theexit plane of liquid crystal cell, depending on the intensities of thebeams.

FIG. 1A schematically illustrates an all-optical Fourier phase-contrastimaging system, according to some embodiments. Laser 110 generates alaser beam with which a phase object is to be irradiated, e.g., a CWbeam from an Ar—Kr laser with a wavelength centered at 480 nm. Ingeneral, the laser wavelength is selected such that the dye in theliquid crystal cell can at least partially absorb the light, and theresulting temperature rise sufficient to induce a phase transitionwithin some of the liquid crystal molecules. Neutral density filter 115adjusts the intensity of the generated laser beam, e.g., in response touser input, in order to adjust the relative phase of the differentspatial frequencies of light in an image of an object being imaged bythe system. Spatial filter 120 includes a pinhole 130 at the focal planeof a microscope objective 125. Spatial filter 120 spatially filters thelaser beam in order to provide a clean, expanded Gaussian profile, andto remove random fluctuations from the intensity profile of the laserbeam, thereby improving the resolution of the imaging system. Othertypes of spatial filters can also be used, such as, for example,diffractive optical elements, beam shapers, and fiber illuminators etc.Lens 140 then collimates the spatially filtered light. As discussed ingreater detail below, beamsplitter 142 and mirror 145 are optional, andcan be used in systems having additional functionalities, such asepifluorescence imaging, as described in greater detail below. Thefiltered light is then directed by beamsplitter 142 and mirror 145 ontospecimen holder 150, which holds the object of interest.

A microscope objective 155, e.g., a 10× microscope objective, collectsthe light transmitted by the object as well as light that did not passthrough the object. The magnification of the image of the object isrelated, in part, to the numerical aperture (NA) of the microscopeobjective 155, which is defined by the half-angle of the cone of lightthat the objective can collect and the index of refraction of the mediumbetween the object of interest and the objective. In general, the higherthe NA of the microscope objective 155, the larger the cone of collectedlight, and thus the more magnified and higher resolution image of theobject can be obtained. Microscope objective 155 is optionally mountedon a motorized x-y-z translation stage. The light transmitted by themicroscope objective 155 is then collimated using a lens (156).

Fourier lens 160, e.g., a bi-convex lens, then performs a Fouriertransform of the light collimated by lens 156. Fourier lens 160 isplaced such that the object or lens 156 is at the front focal plane ofthe lens 160. A liquid crystal cell 165 is placed at the back focalplane of the lens 160. In some embodiments, e.g., embodiments having adye-doped twisted nematic liquid crystal cell, the cell is oriented sothat the incident light is polarized at 45° to the axis of orientationof the liquid crystal. As discussed in greater detail above and below,light in some regions of the Fourier transform of the light accumulate aphase delay relative to light in other regions of the Fourier transformof the light as a result of an intensity-driven phase change. Fourierlens 170 performs an inverse Fourier transformation on the lighttransmitted by liquid crystal cell 165, and images the light onto a CCDarray 180. Fourier lens 170 is placed such that the liquid crystal cell165 is at its front focal plane and the CCD array 180 is at its backfocal plane. CCD array 180 is in communication with a processor 185 thatstores (e.g., in an image store, or a computer-readable medium) orotherwise manipulates the image obtained by CCD array 180 (see above).For example, the processor 185 is in communication with a display device(not shown) on which it displays the resulting phase-contrast image ofthe object.

Optionally, the system includes a polarizer 175 between the Fourier lens170 and the CCD array 180 in order to introduce a self-adaptive spatialfiltering system. Specifically, undesired features of an image can befiltered out by blocking the corresponding spatial frequency componentsat the Fourier plane. In the embodiment of FIG. 1A, the polarizationstate of high spatial frequencies (e.g., regions of liquid crystalphase) is rotated while passing through the liquid crystal cell, whilethere is substantially no such polarization rotation for low spatialfrequencies (e.g., in the isotropic region). Thus, by rotating theanalyzer the desired features of interest can be selectively enhanced,e.g., in order to provide edge enhancement. Hence, for example the(edges) shape of micro organisms can be clearly displayed and even thedimensions can be obtained, e.g., using microscopic rulers.

As noted above, neutral density filter 115 is used to control theincident laser light intensity that illuminates the phase object. Byadjusting the intensity of the laser light, the system illustrated inFIG. 1A can be used in either bright-field imaging mode or phasecontrast imaging mode. Specifically, in bright-field imaging mode, theincident intensity is maintained below the level at which the liquidcrystal phase transition occurs even for low spatial frequencies but ata level that produces a detectible image at the CCD (e.g., from about100 μW to about 10 mW), and the CCD captures a bright-field image. Inphase contrast imaging mode, the incident intensity is increased so thatthe relative phase of some regions of the Fourier transform can bemodified by a phase transition of the liquid crystal, in order toproduce a phase contrast image of the object.

FIG. 1B schematically illustrates the relative phases of light waves asthey travel through different parts of the system of FIG. 1A. Region “A”represents the waves initially transmitted by the laser. In region “A,”substantially all the waves are in phase with each other. As illustratedin region “B,” as the light waves pass through the object on specimenholder 150, some waves get diffracted and/or refracted because of phasegradients (refractive index differences) and accumulate a phase delay,e.g., of π/2. The undeviated waves from those portions of the specimenwhere there is no phase gradient substantially do not accumulate a phasedelay. In the Fourier plane of these waves within liquid crystal cell165, the undeviated light corresponds to low spatial frequenciessituated in the center of the Fourier spectrum, and the deviated lightcorresponds to high spatial frequencies nearer the edges of the Fourierspectrum. The low spatial frequencies at the center of the Fourierspectrum have sufficient intensity to induce a phase change in theliquid crystal cell. In a nematic liquid crystal cell, the low spatialfrequencies are thus located in a region having isotropic phase, whichdoes not have birefringence, and the high spatial frequencies near theedges of the Fourier spectrum are located in a region having liquidcrystal phase with associated birefringence. This causes a phasedifference between high and low spatial frequencies, e.g., of π/2, whichallows the CCD to obtain a phase contrast image of the object.

In one illustrative example, the liquid crystal cell included 90°twisted nematic liquid crystals. The cell walls wereunidirectionally-rubbed poly(vinyl alcohol)-coated glass substrates withthe two directions arranged in a crossed configuration. The substrateswere used to support the polymer film and to hold the liquid crystaltogether. The approximately 10 μm path length cell was filled with auniform mixture of liquid crystal 4-cyno-4′-pentyl1′-1,1′-biphenyl (K15,EM Industries, T_(c)≈35° C.) and absorbing dyeN-ethyl-N-(2-hydroxyethyl)-4-(4-nitrophenylazo) aniline (Disperse Red 1,from Aldrich) which has an absorption peak around 502 nm. In general,any dye concentration providing a temperature increase to cause a phasechange in the liquid crystal in response to a selected laser power,while allowing the cell to transmit sufficient light to produce an imagedetectable at the CCD, can be used. It was observed that typicalincident power required to induce a π/2 phase difference between lightthat passed through the sample, and light that did not, was about 3 mW.

The examples in FIGS. 2A-5C illustrate that some embodiments of systemsand methods of all-optical Fourier phase-contrast imaging usingdye-doped liquid crystals can be used to image biological specimens.Phase contrast images of live amoebae and paramecia include clearlyidentifiable nuclei and other internal organelles. The images equal thequality of images obtained with a standard phase contrast microscope andin some cases display additional features.

FIG. 2A is a bright-field image of a spherical amoeba obtained using thesystem of FIG. 1A in bright-field mode, i.e., at a laser power lowenough to not induce a phase transition in the liquid crystal. Thisbright-field image of the amoeba is a two dimensional structure withpoorly defined edges, and its two larger organelles 210, 211 appear asclouded areas in the center of the specimen. FIG. 2B is a phase contrastimage of a similar amoeba obtained using a conventional phase contrastmicroscope (Leitz Model SM-Lux). This image suggests a partly threedimensional view of the amoeba, and the nucleus 212 and contractilevacuole 213 are more visible than with bright-field microscopy, althoughthey are not sharply focused. Features such as small internal organellesinside the cytoplasm, and edge 214, are more clearly seen than in thecase of the bright-field microscopy image. FIG. 2C is a phase contrastimage of the amoeba of FIG. 2A, obtained using the system of FIG. 1A inphase contrast imaging mode. The nucleus 215, contractile vacuole 216,and smaller organelles that move within the cytoplasm are clearlydefined, and have a visible volume. The image also has a more threedimensional representation of the amoeba than does the conventionalphase microscope image of FIG. 2B, for example, showing multiplepseudopodia 217 at varying depth and in good focus. Phase halos 218,which are one of the drawbacks of a standard phase contrast microscope,can be clearly seen as white outline in FIG. 2B, but are absent in FIG.2C.

FIGS. 3A-3C are images of paramecia, which, like amoebae, are difficultto image using conventional methods because they are they aresubstantially transparent and also frequently move. These unicellularmicroorganisms belong to the protoctist phylum Ciliophora. Members ofthis phylum (ciliates) are characterized by their cigar or slipper shapeand external covering of continuously beating, hair-like cilia. Thesefine structures in particular are not always easy to visualize withbright-field microscopy unless the rest of the specimen is out of focus.Shapes of some of the internal organelles such as a pumping star shapedstructure which constantly expands, contracts, disappears and appears,are typically available only for couple of seconds to take a clearimage.

FIG. 3A is a bright-field image of a paramecium obtained using thesystem of FIG. 1A in bright-field mode, i.e., using a laser power lowenough to not induce a phase transition in the liquid crystal. The imageshows the distinguishing outline 310 and oral groove 311 of theparamecium, but not much else. FIG. 3B is a phase contrast of a similarparamecium obtained using the conventional phase contrast microscope ofFIG. 2B. Details of internal organs 312 can be clearly observed incommercial phase contrast microscope image. FIG. 3C is a phase contrastimage of the paramecium of FIG. 3A, obtained using the system of FIG. 1Ain phase contrast imaging mode. The outline 313 of the paramecium isidentifiable, and the external fine hair-like structures called cilia314 can be seen. The feeding structure, the oral groove, and otherinternal structures are visible in greater detail as compared to FIG.3B.

The system of FIG. 1A can also be used to image a phase object in ascattering medium. FIGS. 4A and 4B are images of translucent onion cellsfrom the skin (peel) of an onion bulb, in a scattering medium. The onionskin was placed in a 2 mm glass cuvette filled with uniform mixture of100 ml of water and 3 ml of Intralipid. Intralipid is widely used inoptical experiments to simulate the scattering properties of biologicaltissues. Solutions of appropriate concentrations of intralipid can beprepared that closely mimic the response of human or animal tissue tolight at wavelengths in the red and infrared ranges, where tissue ishighly scattering but has a rather low absorption coefficient.Kabivitrum Inc., California and Stockholm is a source of Intralipid;there are also other brands (Nutralipid™ (Pharmicia, Quebec), Liposyn™(Abbot Labs, Montreal)) that can be used. Conventionally, solutions ofdistilled water and Intralipid are used as scattering media forbiomedical imaging applications. The mixture simulates the tissueenvironment and matches optical parameters like absorption coefficient,scattering coefficient and the anisotropy coefficient (mean cosine ofthe scattering angle). The reduced scattering coefficient of thesolution is about 6/cm. FIG. 4A is a bright-field image of onion skin inthe scattering medium, obtained using the system of FIG. 1A inbright-field imaging mode. The cell walls 411 are visible and a nucleus410 is noticeable in the picture. FIG. 4B is a phase contrast image ofthe onion skin of FIG. 4A, obtained using the system of FIG. 1A in phasecontrast imaging mode. FIG. 4B shows edges 413 of the cells with muchbetter contrast and the nuclei 412 within are also clearly visible. Astriking feature is that the edge effect is very noticeable in thisimage. It is not possible to obtain phase contrast images for thissample with a standard instrument as it uses an incoherent light source.The high order phase coherence of the coherent source preserves thephase of the scattering medium. However this information is lost when aconventional white light source (incoherent source) is used.

Positive as well as negative phase contrast images can be achieved bysimply varying the intensity of the laser light incident upon thesample. FIGS. 5A-5C are images of a small glass piece that is placed ona micro slide glass. Since the light has to travel through extra glasspiece, it accumulates additional phase as it passes through. FIG. 5A isa bright field image of the glass piece, obtained using the system ofFIG. 1A in bright-field imaging mode. Substantially only the edges 510of the glass piece can be seen. In contrast, phase contrast images suchas illustrated in FIGS. 5B and 5C show variations in optical phaseresulting from transmission through the glass piece. FIG. 5B is apositive phase contrast image, obtained using the system of FIG. 1A inphase contrast imaging mode, FIG. 5C is negative phase contrast imageobtained using the same system but using an incident intensity selectedto provide a −π/2 phase shift between the light passing through theglass piece and the light not passing through the glass piece.

Although phase contrast imaging using 90° twisted nematic liquidcrystals with azobenzene as an absorbing medium is described above,other liquid crystals and other absorbing dyes can also be used. Forexample, zinc 2,9,16,23-tetra-tent-butyl-29H,31H-phthalocyanine as anabsorbing medium and similar phase contrast images were obtained whenthe 648 nm line of Ar—Kr laser is used as pump. Zinc phthalocyanineshave an absorption peak around 677 nm and the liquid crystal cell isprepared in a similar manner as discussed earlier except that the tworubbed substrates are aligned 100 to each other. Broadband dyes can alsobe used, e.g., with a variety of light sources. Thus by selectivelychoosing the absorbing medium, the proposed technique can be used forany wavelength region. For instance 700 nm could be used because it isuseful for in vivo imaging of tissue. Similarly, useful amounts of phasedifference can be achieved by the right combination of birefringence andcell thickness as the phase shift accumulates with length of thebirefringent material.

System arrangements other than those described above can be used toprovide phase contrast imaging using dye doped liquid crystals. Forexample, otherwise conventional microscopes can be modified to havephase contrast imaging capability. FIG. 6 includes a schematicillustration of a conventional microscope 600, along with a photographof an actual conventional microscope. The microscope includes a whitelight source 610, a specimen stage 630, a lens (not shown) between thewhite light source and the specimen stage, a set of interchangeableobjective lenses 640, an eyepiece 650, and a CCD camera 660.

FIG. 7 includes a schematic illustration, as well as a photograph, of amicroscope 700 that includes some of the conventional components of themicroscope of FIG. 6, but performs phase contrast microscopy. Microscope700 includes specimen stage 630 and a set of interchangeable objectivelenses 640, but, instead of a white light source, includes a collimatedlaser source 710, e.g., a diode laser coupled to a fiber collimator.Microscope 700 also includes a phase contrast imaging assembly 760 thatattaches to the body of the conventional microscope, e.g., in place ofCCD 660. Assembly 760 includes Fourier transform lens 761, dye dopedliquid crystal cell 762, Fourier transform lens 763, and CCD array 764.In operation, an objective selected from interchangeable objectivelenses 640 performs an equivalent function to objective 155 in FIG. 1A,i.e., the objective collects light from an object on specimen holder630. Fourier transform lens 761 Fourier transforms the collected light,and the resulting Fourier plane is inside of liquid crystal cell 762,which modifies the relative phases of the transformed light, asdescribed in greater detail above. Fourier transform lens 764 performsan inverse Fourier transform on the light, and images the light onto theCCD 764, thus generating a phase contrast image of the object. CCD 764is in communication with a processor (not shown) that stores (e.g., inan image store, or a computer-readable medium) or otherwise manipulatesthe image obtained by CCD 764 (see above). For example, the processor isin communication with a display device (not shown) on which it displaysthe resulting phase-contrast image of the object.

The systems and methods described above can further be modified toinclude additional functionalities, e.g., that may be complementary tophase contrast imaging. For example, the systems and methods can beadapted to perform other kinds of optical microscopy, such asfluorescence imaging. In contrast to phase contrast microscopy,fluorescence microscopy is capable of imaging the distribution of asingle molecular species based on the properties of its fluorescenceemission. Thus, using fluorescence microscopy, the precise location ofintracellular components labeled with specific fluorophores can bemonitored, for example. Addition of fluorescence imaging capability to aphase contrast microscope allows the system to provide both structuraland functional information.

FIG. 8 illustrates a system 800 that can perform both phase contrastimaging and fluorescence imaging. System 800 is similar in many respectsto the system illustrated in FIG. 1A, and the same components arenumbered with like numbers. System 800 uses a coherent laser source 810,which can be the same or different from the laser source 110 used forphase contrast microscopy. In one example, the laser source is the same,but is tuned to a wavelength that excites a selected fluorophore in theobject to be imaged.

In the illustrated embodiment, signal is obtained from the same side atwhich the object is irradiated, e.g., in an epi-illuminationconfiguration. In this configuration, system 800 includes a mirror 815that directs light transmitted by beamsplitter 142 towards dichroicmirror 820. Dichroic mirror directs light through microscope objective155 to irradiate the object on the specimen holder 155. Fluorescentemission from the object is then captured by microscope objective 155,and transmits through dichroic mirror 820. The emission is then imagedby Fourier lenses 160 and 170 onto CCD array 180. As above, CCD array180 is in communication with a processor 185 that stores (e.g., in animage store, or a computer-readable medium) or otherwise manipulates theimage obtained by CCD array 180 (see above). For example, the processor185 is in communication with a display device (not shown) on which itdisplays the resulting phase-contrast image of the object. The CCD arrayand/or processor may have multi-modal imaging capability.

While the invention has been described in connection with specificmethods and apparatus, those skilled in the art will recognize otherequivalents to the specific embodiments herein. It is to be understoodthat the description is by way of example and not as a limitation to thescope of the invention and these equivalents are intended to beencompassed by the claims set forth below.

1. An assembly for converting a microscope into a phase contrastmicroscope, the microscope comprising a microscope objective and aspecimen stage, wherein the microscope objective is constructed andarranged to collect coherent light from a coherent light source directedtoward the specimen stage, the assembly comprising: a first opticalFourier element that Fourier transforms light from the coherent lightsource, wherein the Fourier transform occurs in a Fourier plane; a cellin the Fourier plane arranged to receive light from the first opticalFourier element, wherein the cell comprises liquid crystal moleculeshaving a phase transition temperature, and wherein at temperaturesexceeding the phase transition temperature, light transmitting throughthe liquid crystal molecules obtains a different phase than lighttransmitting through the liquid crystal molecules at temperatures belowthe phase transition temperature; a second optical Fourier elementarranged to receive light from the cell and inversely Fourier transformthe received light to provide an image; an image sensor that detects theimage and generates an electronic representation of the image; and anadaptor capable of coupling the first and second Fourier elements, thecell, and the image sensor to the microscope such that the first Fourierelement Fourier transforms light collected by the microscope objective.2. The assembly of claim 1, wherein the microscope comprises at leastone of a brightfield and an ordinary microscope.
 3. The assembly ofclaim 1, comprising an optical element configured and arranged to adjustthe light intensity to a level at which part of the transformed lighthas an intensity sufficient to heat a portion of the liquid crystalmolecules above the phase transition temperature.
 4. The assembly ofclaim 3, wherein the optical element selected to adjust the lightintensity comprises a neutral density filter.
 5. The assembly of claim3, wherein the optical element is configured and arranged to adjust thelight intensity to a level at which a portion of the transformed lighttransmits through the cell with a phase that is delayed relative to asecond portion of the transformed light by one of about π/2 and about−π/2.
 6. The assembly of claim 5, wherein the portion of the transformedlight that is phase delayed corresponds to low spatial frequencies, andwherein the second portion of the transformed light corresponds tospatial frequencies that are higher than the portion that is phasedelayed and also has a lower intensity than does the portion that isphase delayed.
 7. The assembly of claim 3, wherein the optical elementis further configured and arranged to adjust the light to an intensitysuch that it does not damage a living organism placed in a sample area.8. The assembly of claim 1, wherein the first and second optical Fourierelements comprise lenses.
 9. The assembly of claim 1, wherein the imagesensor comprises a charge coupled device (CCD).
 10. The assembly ofclaim 1, further comprising a polarizer positioned between the secondoptical Fourier element and the image sensor, the polarizer beingrotatable to a position selected to eliminate at least a part of theinformation about a sample area from the image.
 11. The assembly ofclaim 1, further comprising an optical element to direct at least a partof the light toward a fluorescence imaging system.
 12. The assembly ofclaim 11, wherein the optical element is a dichroic mirror.
 13. Theassembly of claim 1, wherein at temperatures below the phase transitiontemperature, the liquid crystal molecules are birefringent, and whereinat temperatures above the phase transition temperature, the liquidcrystal molecules are isotropic.
 14. The assembly of claim 13, whereinthe light has a predominant polarization, and wherein the liquid crystalcell is oriented at about 45 degrees to the predominant polarization.15. The assembly of claim 1, wherein the liquid crystals in the cellfurther comprises dye molecules selected to at least partially absorbthe light, and wherein at least partial absorption of the light by aportion of the dye molecules heats the part of the liquid crystalmolecules above the phase transition temperature.
 16. The assembly ofclaim 1, wherein below the phase transition temperature, the liquidcrystal molecules are in an aligned nematic phase.
 17. The assembly ofclaim 1, wherein the coherent light source comprises one of acontinuous-wave laser and a diode.
 18. The assembly of claim 1, whereinthe liquid crystal cell is passive, in that no voltage is applied to thecell.
 19. An assembly for converting a microscope into a multi-modalmicroscope, the microscope comprising a microscope objective and aspecimen stage, wherein the microscope objective is constructed andarranged to collect coherent light from a coherent light source directedtoward the specimen stage, the assembly comprising: a first opticalFourier element that Fourier transforms light from the coherent lightsource, wherein the Fourier transform occurs in a Fourier plane; a cellin the Fourier plane arranged to receive light from the first opticalFourier element, wherein the cell comprises liquid crystal moleculeshaving a phase transition temperature, and wherein at temperaturesexceeding the phase transition temperature, light transmitting throughthe liquid crystal molecules obtains a different phase than lighttransmitting through the liquid crystal molecules at temperatures belowthe phase transition temperature; a second optical Fourier elementarranged to receive light from the cell and inversely Fourier transformthe received light to provide an image; an image sensor that detects theimage and generates an electronic representation of the image; anoptical element to direct at least a part of the light toward afluorescence imaging system; and an adaptor capable of coupling thefirst and second Fourier elements, the cell, the image sensor and theoptical element to the microscope such that the first Fourier elementFourier transforms light collected by the microscope objective.
 20. Theassembly of claim 19, wherein the optical element is a dichroic mirror.21. The assembly of claim 20, wherein modes of the multi-modalmicroscope comprise one of at least phase contrast microscopy andfluorescence microscopy.
 22. The assembly of claim 19, wherein thecoherent light source comprises one of a continuous-wave laser and adiode.
 23. A method for converting a microscope into a phase contrastmicroscope, the microscope comprising a microscope objective and aspecimen stage, wherein the microscope objective is constructed andarranged to collect coherent light from a coherent light source directedtoward the specimen stage, the method comprising: providing themicroscope; and coupling a phase contrast assembly to the microscope,the phase contrast assembly comprising: a first optical Fourier elementfor Fourier transforming light from the coherent light source, whereinthe Fourier transforming occurs in a Fourier plane; a cell in theFourier plane arranged for receiving light from the first opticalFourier element, wherein the cell comprises liquid crystal moleculeshaving a phase transition temperature, and wherein at temperaturesexceeding the phase transition temperature, light transmitting throughthe liquid crystal molecules obtains a different phase than lighttransmitting through the liquid crystal molecules at temperatures belowthe phase transition temperature; a second optical Fourier elementarranged for receiving light from the cell and inversely Fouriertransforming the received light to provide an image; an image sensor fordetecting the image and generating an electronic representation of theimage; and an adaptor capable of coupling the first and second Fourierelements, the cell, and the image sensor to the microscope such that thefirst Fourier element Fourier transforms light collected by themicroscope objective.
 24. The method of claim 23, wherein the phasecontrast assembly comprises an optical element for directing at least apart of the light toward a fluorescence imaging system.
 25. The methodof claim 24, wherein the optical element is a dichroic mirror.
 26. Themethod of claim 23, wherein the coherent light source comprises one of acontinuous-wave laser and a diode.
 27. The method of claim 25, whereinthe image sensor comprises a CCD.
 28. A method for converting amicroscope into a multi-modal microscope, the microscope comprising amicroscope objective and a specimen stage, wherein the microscopeobjective is constructed and arranged to collect coherent light from acoherent light source directed toward the specimen stage, the methodcomprising: providing the microscope; and coupling a multi-modalassembly to the microscope, the multi-modal assembly comprising: a firstoptical Fourier element that Fourier transforms light from the coherentlight source, wherein the Fourier transform occurs in a Fourier plane; acell in the Fourier plane arranged to receive light from the firstoptical Fourier element, wherein the cell comprises liquid crystalmolecules having a phase transition temperature, and wherein attemperatures exceeding the phase transition temperature, lighttransmitting through the liquid crystal molecules obtains a differentphase than light transmitting through the liquid crystal molecules attemperatures below the phase transition temperature; a second opticalFourier element arranged to receive light from the cell and inverselyFourier transform the received light to provide an image; an imagesensor that detects the image and generates an electronic representationof the image; an optical element to direct at least a part of the lighttoward a fluorescence imaging system; and an adaptor capable of couplingthe first and second Fourier elements, the cell, the image sensor and anoptical element to the microscope such that the first Fourier elementFourier transforms light collected by the microscope objective.
 29. Themethod of claim 28, wherein the optical element is a dichroic mirror.30. The method of claim 28, wherein modes of the multi-modal microscopecomprise one of phase contrast microscopy and fluorescence microscopy.31. The method of claim 28, wherein the coherent light source comprisesone of a continuous-wave laser and a diode.
 32. A system for convertinga microscope into a phase contrast microscope, the microscope comprisinga microscope objective and a specimen stage, wherein the microscopeobjective is constructed and arranged to collect coherent light directedtoward the specimen stage, the system comprising: a coherent lightsource; and a phase contrast assembly comprising: a first opticalFourier element that Fourier transforms light from the coherent lightsource, wherein the Fourier transform occurs in a Fourier plane; a cellin the Fourier plane arranged to receive light from the first opticalFourier element, wherein the cell comprises liquid crystal moleculeshaving a phase transition temperature, and wherein at temperaturesexceeding the phase transition temperature, light transmitting throughthe liquid crystal molecules obtains a different phase than lighttransmitting through the liquid crystal molecules at temperatures belowthe phase transition temperature; a second optical Fourier elementarranged to receive light from the cell and inversely Fourier transformthe received light to provide an image; an image sensor that detects theimage and generates an electronic representation of the image; and anadaptor capable of coupling the first and second Fourier elements, thecell, and the image sensor to the microscope such that the first Fourierelement Fourier transforms light collected by the microscope objective.33. The system of claim 32, wherein the phase contrast assemblycomprises an optical element to direct at least a part of the lighttoward a fluorescence imaging system.
 34. The system of claim 33,wherein the optical element is a dichroic mirror.
 35. The system ofclaim 32, wherein the coherent light source comprises a continuous-wavelaser and a diode.
 36. A system for converting a microscope into amulti-modal microscope, the microscope comprising a microscope objectiveand a specimen stage, wherein the microscope objective is constructedand arranged to collect coherent light directed toward the specimenstage, the system comprising: a coherent light source; and a multi-modalassembly comprising: a first optical Fourier element that Fouriertransforms light from the coherent light source, wherein the Fouriertransform occurs in a Fourier plane; a cell in the Fourier planearranged to receive light from the first optical Fourier element,wherein the cell comprises liquid crystal molecules having a phasetransition temperature, and wherein at temperatures exceeding the phasetransition temperature, light transmitting through the liquid crystalmolecules obtains a different phase than light transmitting through theliquid crystal molecules at temperatures below the phase transitiontemperature; a second optical Fourier element arranged to receive lightfrom the cell and inversely Fourier transform the received light toprovide an image; an image sensor that detects the image and generatesan electronic representation of the image; an optical element to directat least a part of the light toward a fluorescence imaging system; andan adaptor capable of coupling the first and second Fourier elements,the cell, the image sensor and the optical element to the microscopesuch that the first Fourier element Fourier transforms light collectedby the microscope objective.
 37. The system of claim 36, wherein theoptical element is a dichroic mirror.
 38. The system of claim 37,wherein modes of the multi-modal microscope comprise one of at leastphase contrast microscopy and fluorescence microscopy.
 39. The system ofclaim 36, wherein the coherent light source comprises one of acontinuous-wave laser and a diode.